Implications of a Water Molecule for Photoactivation of Plant (6–4) Photolyase

Abstract

Photolyases (PLs) are flavoproteins able to repair cross-links formed between adjacent pyrimidine bases in DNA in a light-dependent manner via an electron transfer. The catalytically active redox state of the flavin chromophore for the DNA repair is a fully reduced form of flavin adenine dinucleotide (FADH–). PLs and their relative, cryptochromes (CRYs), share a physicochemical process attributable to the light-dependent reduction of the chromophore via an ultrafast successive electron transfer through exclusively conserved three tryptophan side chains. In some (6–4) PLs and animal CRYs, an additional tryptophan participates in this photoactivation process. In a search for the intrinsic difference between the Trp triad and tetrad, a water molecule proximal to the second and third Trp was found in the reported crystal structure of Arabidopsis thaliana (6–4) PL. Here, we investigated the involvement of the water molecule in photoactivation. Molecular dynamics simulations indicated that the water molecule is stably captured in the binding site, while mutation of S412 increased water displacement from the binding site. Photochemical analysis of recombinant proteins revealed that the S412A mutation significantly decelerated the FAD photoreduction as compared to the wild type. The hydrogen-bonding network including the water molecule would play a key role in the stabilization of the FAD–Trp radical pair.

Key interactions with deazariboflavin cofactor for light-driven energy transfer in Xenopus (6–4) photolyase

“…Wild-type Xl64 and Arabidopsis thaliana (6-4)PL (At64) were prepared according to the previous reports using the ampicillin-resistant modified pET28a plasmid encoding either the Xl64 (pET28a'-Xl64) [26] or At64 gene [27], whereas the mutants of Xl64 (F41A, R51A, K256A, and R51A/K256A) were prepared using the kanamycin-resistant pET28a plasmids, which were prepared as mentioned above. Regarding the preparation of the Xl64 mutants, E. coli BL21 (DE3) star cells (Merck) were transformed with the pET28a plasmid bearing the mutant gene.…”

Limited solvation of an electron donating tryptophan stabilizes a photoinduced charge-separated state in plant (6–4) photolyase

“…1 ). Fitting the normalized absorption at 450 nm ( A 450 ) with a monoexponential decay function revealed that FAD photoreduction in At 64-H382S occurred with a half-life ( t 1/2 ) of 222 ± 4 s, which is about tenfold slower than that in At 64-WT 30 ( t 1/2 = 21.6 ± 0.5 s) under the same conditions (Fig. 3 a).…”

“…To examine whether the His/Ser difference affects FAD photoreduction via the Trp-triad, we investigated steady-state photoreduction kinetics for the H382S mutant of At 64 ( At 64-H382S), as previously performed 30 for the wild-type of At 64 ( At 64-WT). At 64-H382S was illuminated by > 430 nm light in the presence of an external reductant under anaerobic conditions and the UV/Vis absorption spectral changes were monitored.…”

“…3 a). To examine whether the less efficient photoreduction caused by the H382S mutation affects the (6–4)PP photorepair activity of At 64, UV-sensitive E. coli SY32 cells, in which CPD lesions can be repaired due to a rescue plasmid coding E. coli CPD PL gene, were transformed with an At 64-H382S expressing plasmid, in the same way as previously reported 30 . The survival rate was found to be about tenfold lower in H382S than in WT (Fig.…”

“…Given the observations that FAD photoreduction in At 64 significantly slowed down upon mutation of His382 to relatively compact and polar residues (Ser, Asn, and Asp), we considered the possible involvement of solvation of Trp 3 H in the photoreduction. To evaluate the solvation, we performed molecular dynamics (MD) simulations for the His382 mutants in the same way as previously reported 30 , and analyzed the presence of water molecules around the Trp 3 H in the last 100 ns of the production runs. We defined the area within 3.4 and 5.0 Å of the nitrogen atom of the Trp 3 H indole ring as the first (HS1) and the second (HS2) hydration shell, respectively (Fig.…”